The present invention relates to a method and test composition for the determination of hydrogen peroxide, and more particularly, to a method for the determination of hydrogen peroxide by reacting hydrogen peroxide with a novel chromogen as a hydrogen donor in the presence of peroxidase and determining the degree of pigment formed. The invention also pertains to a test composition suitable for carrying out such determination.
Heretofore, the determination of a substrate is generally carried out by oxidizing the substrate by the action of oxidase and determining the formed hydrogen peroxide. For example, cholesterol is oxidized by cholesterol oxidase to form hydrogen peroxide. The hydrogen peroxide is then determined by reacting the hydrogen peroxide with a chromogen in the presence of peroxidase to form a pigment and measuring the absorbancy of the reaction solution colored by the formation of the pigment in the visible ray region. In such processes, 4-amino-antipyrine (hereinafter referred to as "4AA") and phenol, 4AA and N,N-dimethylaniline, 4AA and N-ethyl-N-(.beta.-hydroxyethyl)-m-toluidine, 3-methylbenzothiazolin hydrazone and N,N-diethylaniline, 4AA and N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine (hereinafter referred to as "EMAE") and the like are generally used as the chromogen.
The pigments formed by using these chromogens lacks stability under alkaline sides beyond pH 7.0.
On the other hand, the optimum pH of many enzymes used for diagnostic purposes is on the alkaline side.
Therefore, there is a need to develop chromogens which are superior in sensitivity, and stable on the alkaline side.